Merkel cell polyomavirus (MCPyV) is a ubiquitous skin infection that can cause Merkel cell carcinoma (MCC), a highly lethal form of skin cancer with a nearly 50% mortality rate. Since the discovery of MCPyV in 2008, great advances have been made to improve our understanding of how the viral encoded oncoproteins contribute to MCC oncogenesis. However, our knowledge of the MCPyV infectious life cycle and its oncogenic mechanisms are still incomplete. The incidence of MCC has tripled over the past two decades, but effective treatments are lacking. Only recently have there been major victories in combatting metastatic MCC with the application of PD-1 immune checkpoint blockade. Still, these immune-based therapies are not ideal for patients with a medical need to maintain systemic immune suppression. As such, a better understanding of MCPyV's oncogenic mechanisms is needed in order to develop more effective and targeted therapies against virus-associated MCC. In this review, we discuss current areas of interest for MCPyV and MCC research and the progress made in elucidating both the natural host of MCPyV infection and the cell of origin for MCC. We also highlight the remaining gaps in our knowledge on the transcriptional regulation of MCPyV, which may be key to understanding and targeting viral oncogenesis for developing future therapies.

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post-integration (~80% cases), or by UV-mediated DNA damage. Interestingly, overall survival of MCV-positive Merkel cell carcinoma patients is better, making this differential information of significant diagnostic and prognostic value. Also, MCV provides a direct target for therapy in MCC patients. Currently, the methods used for diagnosis of MCV in tumors are often discordant and unreliable. Here we used a guided molecular scissors based-DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an in vitro molecular diagnostic tool for MCV-positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme-gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that MCV DETECTR system can detect MCV integrated in Merkel tumor rapidly, specifically and with femto-molar sensitivity. Our study is a preliminary, proof-of-principle analysis showing the use of CRISPR for MCV diagnosis. Further validation in human tumor samples is needed for its clinical use in the near future. This new system is promising and we hope it can be coupled with immunohistochemical studies to diagnose the viral status of MCC in clinics soon.

Merkel cell polyomavirus (MCPyV) is a small DNA virus with oncogenic potential. MCPyV is the causative agent of Merkel Cell Carcinoma (MCC), a rare but aggressive tumor of the skin. The role of epigenetic mechanisms, such as histone posttranslational modifications (HPTMs), DNA methylation, and microRNA (miRNA) regulation on MCPyV-driven MCC has recently been highlighted. In this review, we aim to describe and discuss the latest insights into HPTMs, DNA methylation, and miRNA regulation, as well as their regulative factors in the context of MCPyV-driven MCC, to provide an overview of current findings on how MCPyV is involved in the dysregulation of these epigenetic processes. The current state of the art is also described as far as potentially using epigenetic dysregulations and related factors as diagnostic and prognostic tools is concerned, in addition to targets for MCPyV-driven MCC therapy. Growing evidence suggests that the dysregulation of HPTMs, DNA methylation, and miRNA pathways plays a role in MCPyV-driven MCC etiopathogenesis, which, therefore, may potentially be clinically significant for this deadly tumor. A deeper understanding of these mechanisms and related factors may improve diagnosis, prognosis, and therapy for MCPyV-driven MCC.

Merkel cell polyomavirus (MCPyV) has been detected in approximately 75% of patients with the rare skin cancer Merkel cell carcinoma. We investigated the prevalence of antibodies against MCPyV in the general population and the association between these antibodies and Merkel cell carcinoma.

Merkel cell polyomavirus (MCPyV) is associated to Merkel cell carcinoma (MCC). We studied 113 MCC tumoral skin lesions originating from 97 patients. MCPyV detection was higher in fresh-frozen (FF) biopsies (94%) than in formalin-fixed paraffin-embedded biopsies (39-47%). Mean viral load in FF tumor was of 7.5 copies per cell with a very wide range (0.01-95.4). Nineteen complete sequences of LTAg were obtained, mainly from FF biopsies when the viral load was high. Seventeen showed stop codons, all localized downstream of the pRb protein binding domain. Sequence comparison and phylogenetic analysis showed that all sequences clustered in the large C clade of MCPyV strains. MCPyV integration was demonstrated in 19 out of 27 FF MCC DNA biopsies without evidence of specific host cellular genome integration site. In 13/19 cases, the viral junction was located within the second exon of the LTAg, after the pRB binding domain.

The surface of polyomavirus virions is composed of pentameric knobs of the major capsid protein, VP1. In previously studied polyomavirus species, such as SV40, two interior capsid proteins, VP2 and VP3, emerge from the virion to play important roles during the infectious entry process. Translation of the VP3 protein initiates at a highly conserved Met-Ala-Leu motif within the VP2 open reading frame. Phylogenetic analyses indicate that Merkel cell polyomavirus (MCV or MCPyV) is a member of a divergent clade of polyomaviruses that lack the conserved VP3 N-terminal motif. Consistent with this observation, we show that VP3 is not detectable in MCV-infected cells, VP3 is not found in native MCV virions, and mutation of possible alternative VP3-initiating methionine codons did not significantly affect MCV infectivity in culture. In contrast, VP2 knockout resulted in a >100-fold decrease in native MCV infectivity, despite normal virion assembly, viral DNA packaging, and cell attachment. Although pseudovirus-based experiments confirmed that VP2 plays an essential role for infection of some cell lines, other cell lines were readily transduced by pseudovirions lacking VP2. In cell lines where VP2 was needed for efficient infectious entry, the presence of a conserved myristoyl modification on the N-terminus of VP2 was important for its function. The results show that a single minor capsid protein, VP2, facilitates a post-attachment stage of MCV infectious entry into some, but not all, cell types.

To clarify whether mutations in the large T gene encoded by Merkel cell polyomavirus affect the expression and function of large T antigen in Merkel cell carcinoma cases, we investigated the expression of large T antigen in vitro and in vivo. Immunohistochemistry using a rabbit polyclonal antibody revealed that large T antigen was expressed in the nuclei of Merkel cell carcinoma cells with Merkel cell polyomavirus infection. Deletion mutant analyses identified an Arg-Lys-Arg-Lys sequence (amino acids 277-280) as a nuclear localization signal in large T antigen. Sequence analyses revealed that there were no mutations in the nuclear localization signal in any of the eleven Merkel cell polyomavirus strains examined. Furthermore, stop codons were not observed in the upstream of the nuclear localization signal in any of the Merkel cell carcinoma cases examined. These data suggest that the nuclear localization signal is highly conserved and functional in Merkel cell carcinoma cases.

Merkel cell carcinoma (MCC) is a rare, high-grade, aggressive cutaneous neuroendocrine malignancy most commonly associated with sun-exposed areas of older individuals. A relatively newly identified human virus, the Merkel cell polyomavirus (MCPyV) has been implicated in the pathogenesis of MCC. Our study aimed to examine nine MCC cases and randomly selected 60 melanoma cases to identify MCPyV status and to elucidate genetic differences between virus-positive and -negative cases. Altogether, seven MCPyV-positive MCC samples and four melanoma samples were analyzed. In MCPyV-positive MCC RB1, TP53, FBXW7, CTNNB1, and HNF1A pathogenic variants were identified, while in virus-negative cases only benign variants were found. In MCPyV-positive melanoma cases, besides BRAF mutations the following genes were also affected: PIK3CA, STK11, CDKN2A, SMAD4, and APC. In contrast to studies found in the literature, a higher tumor burden was detected in virus-associated MCC compared to MCPyV-negative cases. No association was identified between virus infection and tumor burden in melanoma samples. We concluded that analyzing the key morphologic and immunohistological features of MCC is critical to avoid confusion with other cutaneous malignancies. Molecular genetic investigations such as next-generation sequencing (NGS) enable molecular stratification, which may have future clinical impact.

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

In 2008, Feng et al. identified Merkel cell polyomavirus integration as the primary oncogenic event in ~80% of Merkel cell carcinoma cases. The remaining virus-negative Merkel cell carcinoma cases associated with a high mutational load are most likely caused by UV radiation. The current study aimed to compare the morphological and immunohistochemical features of 80 virus-positive and 21 virus-negative Merkel cell carcinoma cases. Microscopic evaluation revealed that elongated nuclei-similar to the spindle-shape variant of small cell lung cancer-were less frequent in Merkel cell polyomavirus-positive Merkel cell carcinoma compared to the virus-negative subset (p = 0.005). Moreover, virus-negative cases more frequently displayed a "large-cell neuroendocrine carcinoma" phenotype with larger cell size (p = 0.0026), abundant cytoplasm (p = 4×10-7) and prominent nucleoli (p = 0.002). Analysis of immunohistochemical data revealed frequent positivity for thyroid transcription factor 1 and cytokeratin 7, either absence or overexpression of p53, as well as frequent lack of neurofilament expression in virus-negative cases. By contrast, cytokeratin 8, 18 and 20 and a CD99 with a dot pattern as well as high EMA expression were identified as characteristic features of virus-positive Merkel cell carcinoma. In particular, the CD99 dot-like expression pattern was strongly associated with presence of the Merkel cell polyomavirus in Merkel cell carcinoma (sensitivity = 81%, specificity = 90%, positive likelihood ratio = 8.08). To conclude, virus-positive and -negative Merkel cell carcinoma are characterized by distinct morphological and immunohistochemical features, which implies a significant difference in tumor biology and behavior. Importantly, we identified the CD99 staining pattern as a marker indicating the virus status of this skin cancer.

Merkel cell carcinoma (MCC) is a rare but aggressive form of skin cancer predominantly caused by the human Merkel cell polyomavirus (MCPyV). Treatment for MCC includes excision and radiotherapy of local disease, and chemotherapy or immunotherapy for metastatic disease. The schweinfurthin family of natural compounds previously displayed potent and selective growth inhibitory activity against the NCI-60 panel of human-derived cancer cell lines. Here, we investigated the impact of schweinfurthin on human MCC cell lines. Treatment with the schweinfurthin analog, 5'-methylschweinfurth G (MeSG also known as TTI-3114), impaired metabolic activity through induction of an apoptotic pathway. MeSG also selectively inhibited PI3K/AKT and MAPK/ERK pathways in the MCPyV-positive MCC cell line, MS-1. Interestingly, expression of the MCPyV small T (sT) oncogene selectively sensitizes mouse embryonic fibroblasts to MeSG. These results suggest that the schweinfurthin family of compounds display promising potential as a novel therapeutic option for virus-induced MCCs.

Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, β-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.

Seborrheic keratosis (SK) is the most common benign cutaneous neoplasm. A subset shows increased p16 expression. Since SK shares several features with verruca vulgaris, e.g., increased p16 expression, human papillomaviruses (HPV) have been suggested as possible causal agents. However, a relevant association could not be established between HPV and SK. In the present study we aimed to investigate the presence of Merkel cell polyomavirus (MCPyV) in relation to p16 expression in SK. P16 expression was investigated using immunohistochemistry (IHC). Presence of MCPyV was assessed in 23 formalin-fixed paraffin-embedded tissue samples of SK by molecular techniques (i.e., PCR and FISH) and IHC. 16/23 SK showed strong to moderate p16 expression. 6/23 of SK were MCPyV positive by PCR which was confirmed by FISH. Of interest, two samples with strong FISH signals also showed MCPyV expression as tested by IHC. Samples with weaker signal intensity were negative in IHC. P16 expression was not associated with the presence of MCPyV. Concluding, the detection of MCPyV DNA by PCR and FISH in SK reflects the widespread prevalence of MCPyV in the skin. However, low detection rates exclude MCPyV as a major pathogenic factor in SK, most likely representing a coincidental infection. P16 IHC does not appear as useful adjunctive surrogate marker for the presence of MCPyV in SK.

Merkel cell carcinoma (MCC) is an aggressive and lethal type of neuroendocrine skin cancer. Mutated Merkel cell polyomavirus (MCV) is commonly found in MCC, and leads to upregulation of the survivin oncogene. However, ∼20% of MCC tumors do not have detectable MCV, suggesting alternative etiologies for this tumor type. In this study, our aim was to evaluate microRNA (miRNA) expression profiles and their associations with MCV status and clinical outcomes in MCC. We showed that miRNA expression profiles were distinct between MCV-positive (MCV+) and MCV-negative (MCV-) MCCs and further validated that miR-203, miR-30a-3p, miR-769-5p, miR-34a, miR-30a-5p, and miR-375 were significantly different. We also identified a subset of miRNAs associated with tumor metastasis and MCC-specific survival. Functionally, overexpression of miR-203 was found to inhibit cell growth, induce cell cycle arrest, and regulate survivin expression in MCV- MCC cells, but not in MCV+ MCC cells. Our findings reveal a mechanism of survivin expression regulation in MCC cells, and provide insights into the role of miRNAs in MCC tumorigenesis.

Merkel cell polyomavirus (MCPyV) is the etiological agent of the majority of Merkel cell carcinoma (MCC): a rare skin tumor. To improve our understanding of the role of MCPyV in MCCs, the detection and analysis of MCPyV DNA and transcripts were performed on primary tumors and regional lymph nodes from two MCC patients: one metastatic and one non-metastatic. MCPyV-DNA was searched by a quantitative polymerase chain reaction (qPCR), followed by the amplification of a Large T Antigen (LTAg), Viral Protein 1 (VP1) and Non-Coding Control Region (NCCR). LTAg and VP1 transcripts were investigated by reverse-transcription PCR (RT-PCR). Viral integration was also studied, and full-length LTAg sequencing was performed. qPCR revealed that the primary tumor of both patients and the lymph node of one patient was positive for the small t-antigen, with an average value of 7.0 × 102 copies/µg. The same samples harbored LTAg, NCCR and VP1 DNA. Sequencing results showed truncated LTAg with the conserved retinoblastoma (Rb) protein binding motif and VP1 and NCCR sequences identical to the MCC350 strain. RT-PCR detected LTAg but not VP1 transcripts. The MCPyV genome was integrated into the primary tumor of both patients. The results confirmed the connection between MCPyV and MCC, assuming integration, LTAg truncation and Rb sequestration as key players in MCPyV-mediated oncogenesis.

Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). It is still under discussion, in which cells viral integration and MCC development occurs. Recently, we demonstrated that a virus-positive MCC derived from a trichoblastoma, an epithelial neoplasia bearing Merkel cell (MC) differentiation potential. Accordingly, we hypothesized that MC progenitors may represent an origin of MCPyV-positive MCC. To sustain this hypothesis, phenotypic comparison of trichoblastomas and physiologic human MC progenitors was conducted revealing GLI family zinc finger 1 (GLI1), Keratin 17 (KRT 17), and SRY-box transcription factor 9 (SOX9) expressions in both subsets. Furthermore, GLI1 expression in keratinocytes induced transcription of the MC marker SOX2 supporting a role of GLI1 in human MC differentiation. To assess a possible contribution of the MCPyV T antigens (TA) to the development of an MC-like phenotype, human keratinocytes were transduced with TA. While this led only to induction of KRT8, an early MC marker, combined GLI1 and TA expression gave rise to a more advanced MC phenotype with SOX2, KRT8, and KRT20 expression. Finally, we demonstrated MCPyV-large T antigens' capacity to inhibit the degradation of the MC master regulator Atonal bHLH transcription factor 1 (ATOH1). In conclusion, our report suggests that MCPyV TA contribute to the acquisition of an MC-like phenotype in epithelial cells.

Merkel cell carcinoma (MCC) is a relatively uncommon but highly lethal form of skin cancer. A majority of MCC tumors carry DNA sequences derived from a newly identified virus called Merkel cell polyomavirus (MCV or MCPyV), a candidate etiologic agent underlying the development of MCC. To further investigate the role of MCV infection in the development of MCC, we developed a reporter vector-based neutralization assay to quantitate MCV-specific serum antibody responses in human subjects. Our results showed that 21 MCC patients whose tumors harbored MCV DNA all displayed vigorous MCV-specific antibody responses. Although 88% (42/48) of adult subjects without MCC were MCV seropositive, the geometric mean titer of the control group was 59-fold lower than the MCC patient group (p<0.0001). Only 4% (2/48) of control subjects displayed neutralizing titers greater than the mean titer of the MCV-positive MCC patient population. MCC tumors were found not to express detectable amounts of MCV VP1 capsid protein, suggesting that the strong humoral responses observed in MCC patients were primed by an unusually immunogenic MCV infection, and not by viral antigen expressed by the MCC tumor itself. The occurrence of highly immunogenic MCV infection in MCC patients is unlikely to reflect a failure to control polyomavirus infections in general, as seroreactivity to BK polyomavirus was similar among MCC patients and control subjects. The results support the concept that MCV infection is a causative factor in the development of most cases of MCC. Although MCC tumorigenesis can evidently proceed in the face of effective MCV-specific antibody responses, a small pilot animal immunization study revealed that a candidate vaccine based on MCV virus-like particles (VLPs) elicits antibody responses that robustly neutralize MCV reporter vectors in vitro. This suggests that a VLP-based vaccine could be effective for preventing the initial establishment of MCV infection.

Merkel cell carcinoma is a rare, lethal cancer histopathologically composed of cells showing similarity with mechanoreceptor Merkel cells. Merkel cell tumors manifest in two distinct forms. While a virus called Merkel cell polyomavirus is involved in the pathogenesis of one form of Merkel tumors, the other is driven by ultraviolet (UV)-linked mutations. In this study we investigated 18 cases, from the Indian population, of Merkel cell carcinoma for immunohistochemical (IHC) expression of Merkel cell polyomavirus (MCV) T antigen, including 12 cases tested by PCR, to identify viral etiopathology. We tested the tumors with two sensitive antibodies (CM2B4 and Ab3), targeting the viral large T antigen protein and with PCR primers targeting the N terminus of T antigen. Overall, we observed 38.8% (7/18) tumors displaying positive IHC expression of Merkel cell polyomavirus T antigen and 25% (3/12) tumors showing positive results, by both, immunohistochemistry and PCR. This constitutes the first report from India showing implication of MCV in Merkel cell carcinomas. Moreover, this is one of the larger series of Merkel cell carcinomas, tested for MCV, by both immunohistochemistry and PCR, in this part of the world. These results further indicate that a slightly more number of such cases in India are likely to be caused by UV-linked damage, as opposed to Merkel cell polyomavirus mediated tumorigenesis, which is definitely implicated in a subset of cases.

Merkel cell polyomavirus (MCPyV) is a human polyomavirus that establishes a life-long harmless infection in most individuals, with dermal fibroblasts believed to be the natural host cell. However, this virus is the major cause of Merkel cell carcinoma (MCC), an aggressive skin cancer. Several MCPyV variants with polymorphism in their promoter region have been isolated, but it is not known whether these differences affect the biological properties of the virus.

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.